miR-661 downregulates both Mdm2 and Mdm4 to activate p53

The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.     

抑癌因子ARF的非p53调节通路

ARF蛋白是INK4a基因位点编码产物之一,是一种重要的肿瘤抑制因子。ARF可结合原癌蛋白Mdm2,稳定p53,将细胞周期阻断在G1期和G2/M转换期,或诱导细胞凋亡。有关ARF的p53依赖性作用已有较多报道。该文主要以ARF对E2F1、DP1、E2F1/DP1、NPM/B23和c-Myc等的调控为例,对ARF的非p53调节通路做一综述。     

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.     

Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.     

PIASy-mediated Tip60 sumoylation regulates p53-induced autophagy

Posttranslational modifications of p53 integrate diverse stress signals and regulate its activity, but their combinatorial contribution to overall p53 function is not clear. We investigated the roles of lysine (K) acetylation and sumoylation on p53 and their relation to apoptosis and autophagy. Here we describe the collaborative role of the SUMO E3 ligase PIASy and the lysine acetyltransferase Tip60 in p53-mediated autophagy. PIASy binding to p53 and PIASy-activated Tip60 lead to K386 sumoylation and K120 acetylation of p53, respectively. Even though these two modifications are not dependent on each other, together they act as a “binary death signal” to promote cytoplasmic accumulation of p53 and execution of PUMA-independent autophagy. PIASy-induced Tip60 sumoylation augments p53 K120 acetylation and apoptosis. In addition to p14^ARF inactivation, impairment in this intricate signaling may explain why p53 mutations are not found in nearly 50% of malignancies.     

PIASy-mediated Tip60 sumoylation regulates p53-induced autophagy

Posttranslational modifications of p53 integrate diverse stress signals and regulate its activity, but their combinatorial contribution to overall p53 function is not clear. We investigated the roles of lysine (K) acetylation and sumoylation on p53 and their relation to apoptosis and autophagy. Here we describe the collaborative role of the SUMO E3 ligase PIASy and the lysine acetyltransferase Tip60 in p53-mediated autophagy. PIASy binding to p53 and PIASy-activated Tip60 lead to K386 sumoylation and K120 acetylation of p53, respectively. Even though these two modifications are not dependent on each other, together they act as a “binary death signal” to promote cytoplasmic accumulation of p53 and execution of PUMA-independent autophagy. PIASy-induced Tip60 sumoylation augments p53 K120 acetylation and apoptosis. In addition to p14^ARF inactivation, impairment in this intricate signaling may explain why p53 mutations are not found in nearly 50% of malignancies.     

Mdmx enhances p53 ubiquitination by altering the substrate preference of the Mdm2 ubiquitin ligase

mdm2 and mdmx oncogenes play essential yet non-redundant roles in synergistic inactivation of the tumor suppressor, p53. While Mdm2 inhibits p53 activity mainly by augmenting its ubiquitination, the functional role of Mdmx on p53 ubiquitination remains obscure. In transfected H1299 cells, Mdmx augmented Mdm2-mediated ubiquitination of p53. In in vitro ubiquitination assays, the Mdmx/Mdm2 heteromeric complex, in comparison to the Mdm2 homomer, showed enhanced ubiquitinase activity toward p53 and the reduced auto-ubiquitination of Mdm2. Alteration of the substrate specificity via binding to Mdmx may contribute to efficient ubiquitination and inactivation of p53 by Mdm2.

Structured summary

MINT-7219995: P53 (uniprotkb:P04637) physically interacts (MI:0914) with Ubiquitin (uniprotkb:P62988) by anti bait coimmunoprecipitation (MI:0006)MINT-7220023: Ubiquitin (uniprotkb:P62988) physically interacts (MI:0914) with P53 (uniprotkb:P04637) by pull down (MI:0096)     

KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or gamma-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.     

p53-Mdm2模块和泛素化系统

p53(肿瘤抑制基因)诱导鼠双微粒体蛋白2(Mdm2)的表达,Mdm2反之抑制p53的活性,Mdm2和p53形成了一个自动调整的模块。Mdm2的一个重要的结构标志是一个中心酸性区域,另外的结构标志是在酸结构域下游的一个锌指结构,和一个C端的环指区域。Mdm2的表达是由p53来调节,Mdm2作为E3连接酶使p53泛素化并且驱使p53降解,进而控制p53的功能。对于p53泛素化的结构要求是p53的寡聚化。p53泛素化作用的调整模式是通过蛋白质之间的相互作用。Mdm2中环指区域的作用是通过使p53泛素化来推进p53的降解。泛素化后的酸性结构在Mdm2的降解中起作用。     

HAUSP-regulated switch from auto- to p53 ubiquitination by Mdm2 (in silico discovery)

Stability of the 'guardian of the genome' tumor suppressor protein p53 is regulated predominantly through its ubiquitination. The ubiquitin-specific protease HAUSP plays an important role in this process. Recent experiments showed that p53 demonstrates a differential response to changes in HAUSP which nature and significance are not understood yet. Here a data-driven mathematical model of the Mdm2-mediated p53 ubiquitination network is presented which offers an explanation for the cause of such a response. The model predicts existence of the HAUSP-regulated switch from auto- to p53 ubiquitination by Mdm2. This switch suggests a potential role of HAUSP as a downstream target of stress signals in cells. The model accounts for a significant amount of experimental data, makes predictions for some rate constants, and can serve as a building block for the larger model describing a complex dynamic response of p53 to cellular stresses.     

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21^Cip1 and p27^Kip1. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.     

Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopy

Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour‐suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down‐regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N‐terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein–protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti‐cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd.     

Regulation of the Mdm2–p53 pathway by the ubiquitin E3 ligase MARCH7

The tumor suppressor p53 plays a prominent role in the protection against cancer. The activity of p53 is mainly controlled by the ubiquitin E3 ligase Mdm2, which targets p53 for proteasomal degradation. However, the regulation of Mdm2 remains not well understood. Here, we show that MARCH7, a RING domain‐containing ubiquitin E3 ligase, physically interacts with Mdm2 and is essential for maintaining the stability of Mdm2. MARCH7 catalyzes Lys⁶³‐linked polyubiquitination of Mdm2, which impedes Mdm2 autoubiquitination and degradation, thereby leading to the stabilization of Mdm2. MARCH7 also promotes Mdm2‐dependent polyubiquitination and degradation of p53. Furthermore, MARCH7 is able to regulate cell proliferation, DNA damage‐induced apoptosis, and tumorigenesis via a p53‐dependent mechanism. These findings uncover a novel mechanism for the regulation of Mdm2 and reveal MARCH7 as an important regulator of the Mdm2–p53 pathway.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

TRIM29 negatively regulates p53 via inhibition of Tip60

Ataxia-telangiectasia (AT) is an autosomal recessive genetic disease characterized by immunological deficiencies, neurological degeneration, developmental abnormalities and an increased risk of cancer. Ataxia-telangiectasia group D (ATDC) was initially described as a gene related to AT. Ataxia-telangiectasia group D, also known as TRIM29, is structurally a member of the tripartite motif (TRIM) family of proteins, some of which have been reported to be highly expressed in some human carcinomas, but the involvement of TRIM29 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM29 binds to Tip60, which has been reported as a cellular acetyltransferase protein. Overexpression of TRIM29 promoted degradation and changed localization of Tip60 and reduced acetylation of p53 at lysine 120 by Tip60, resulting in enhancement of cell growth and transforming activity. In addition, we found that TRIM29 suppresses apoptosis induced by UV irradiation in HCT116 cell lines. These findings suggest that TRIM29 functions as an oncogene that promotes tumor growth.